Zhang Shang-Min, Chen Kun-Ming, Sun Yuan-Wan, Aliaga Cesar, Lin Jyh-Ming, Sharma Arun K, Amin Shantu, El-Bayoumy Karam
Dept. of Biochemistry and Molecular Biology, Penn State College of Medicine , Hershey, Pennsylvania 17033, United States.
Chem Res Toxicol. 2014 Jul 21;27(7):1199-206. doi: 10.1021/tx5001078. Epub 2014 Jun 17.
We were the first to demonstrate that direct application of the environmental pollutant and tobacco smoke constituent dibenzo[a,l]pyrene (DB[a,l]P) into the oral cavity of mice induced squamous cell carcinoma (SCC) in oral tissues but not in the tongue; however, the mechanisms that can account for the varied carcinogenicity remain to be determined. Furthermore, we also showed that not only dA adducts, but also dG adducts can account for the mutagenic activity of DB[a,l]P in the oral tissues in vivo. In this study, we initially focused on DB[a,l]P-induced genotoxic effects in both oral and tongue tissues. Therefore, to fully assess the contribution of these DNA adducts in the initiation stage of carcinogenesis induced by DB[a,l]P, an LC-MS/MS method to simultaneously detect and quantify DB[a,l]PDE-dG and -dA adducts was developed. Mice were orally administered with DB[a,l]P (24 nmole, 3 times per week for 5 weeks) or its fjord region diol epoxide, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE, 12 nmole, single application); animals were sacrificed at 2, 7, 14, and 28 days after the last dose of carcinogen administration. Oral and tongue tissues were obtained and DNA were isolated followed by enzymatic hydrolysis. Following the development of an isotope dilution LC-MS/MS method, we successfully detected (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(2)-dG, as well as (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(6)-dA in oral and tongue tissues of mice treated with DB[a,l]P. Levels of (-)-anti-trans-DB[a,l]PDE-N(6)-dA were ≥2 folds higher than (-)-anti-cis-DB[a,l]PDE-N(6)-dA adduct and those of dG adducts in the oral tissues and tongue at all time points selected after the cessation of DB[a,l]P treatment. Levels of dG adducts were comparable in both tissues. Collectively, our results support that DB[a,l]P is predominantly metabolized to (-)-anti-DB[a,l]PDE, and the levels and persistence of (-)-anti-trans-DB[a,l]PDE-N(6)-dA may, in part, explain the carcinogenicity of DB[a,l]P in the oral tissues but not in the tongue.
我们首次证明，将环境污染物及烟草烟雾成分二苯并[a,l]芘（DB[a,l]P）直接施用于小鼠口腔可诱导口腔组织发生鳞状细胞癌（SCC），但舌部未发生；然而，导致这种不同致癌性的机制仍有待确定。此外，我们还表明，不仅dA加合物，dG加合物也可解释DB[a,l]P在体内口腔组织中的诱变活性。在本研究中，我们最初聚焦于DB[a,l]P在口腔和舌组织中诱导的基因毒性效应。因此，为了全面评估这些DNA加合物在DB[a,l]P诱导的致癌起始阶段的作用，我们开发了一种液相色谱-串联质谱（LC-MS/MS）方法，用于同时检测和定量DB[a,l]PDE-dG和-dA加合物。给小鼠口服DB[a,l]P（24纳摩尔，每周3次，共5周）或其峡湾区二醇环氧化物（±）-反式-11,12-二羟基-13,14-环氧-11,12,13,14-四氢二苯并[a,l]芘（DB[a,l]PDE，12纳摩尔，单次给药）；在最后一次给予致癌物后2、7、14和28天处死动物。获取口腔和舌组织并分离DNA，随后进行酶水解。在开发出同位素稀释LC-MS/MS方法后，我们成功在经DB[a,l]P处理的小鼠口腔和舌组织中检测到（-）-反式-顺式和（-）-反式-反式-DB[a,l]PDE-N(2)-dG，以及（-）-反式-顺式和（-）-反式-反式-DB[a,l]PDE-N(6)-dA。在停止DB[a,l]P处理后所选的所有时间点，口腔组织和舌部中（-）-反式-DB[a,l]PDE-N(6)-dA的水平比（-）-反式-顺式-DB[a,l]PDE-N(6)-dA加合物及dG加合物的水平高≥2倍。两种组织中dG加合物的水平相当。总体而言，我们的结果支持DB[a,l]P主要代谢为（-）-反式-DB[a,l]PDE，且（-）-反式-DB[a,l]PDE-N(6)-dA的水平和持久性可能部分解释了DB[a,l]P在口腔组织而非舌部的致癌性。
